Monoglyceride Lipase and Hormone-Sensitive Lipase - Molecular and structural aspects

Sammanfattning: Triglycerides in the adipocyte are hydrolysed by means of three consecutive reactions through the combined action of two lipases: hormone-sensitive lipase (HSL) and monoglyceride lipase (MGL). HSL catalyses the first and rate-limiting step, i.e. the hydrolysis of triglycerides to diglycerides, and the subsequent hydrolysis of diglycerides and monoglycerides. However, MGL is needed to assure complete hydrolysis of the monoglycerides. HSL is subjected to acute hormonal and neural control through reversible phosphorylation, whereas MGL presumably lacks any kind of short-term regulation. The cDNAs encoding mouse and human adipose tissue MGL were cloned. The mouse MGL protein is predicted to consist of 302 amino acids and to have a molecular weight of 33,218. Two different 5’ leader sequences were found to be present in adipocytes. Northern blot analyses of rat and human tissues suggest that MGL is ubiquitously expressed with a transcript size of approximately 4 kb. This size is in agreement with the cloned cDNA sequences. Western blot analysis revealed heterogeneities, both in number and length of MGL proteins, in various mouse tissues. The amino acid sequence identity between mouse and human MGL is 84%. The structural elements and the putative catalytic triads in both HSL and MGL were identified by means of sequence alignments and secondary structure predictions. Both enzymes were found to exhibit the alfa/beta hydrolase fold that is characteristic of lipases. The residues of the proposed triad in MGL were confirmed by site-directed mutagenesis. Using the baculovirus-insect cell system, mouse MGL was produced as a fusion protein with an amino-terminal stretch of six histidines. Purification to apparent homogeneity was obtained by nickel-chelating chromatography. Characterisation of the mouse MGL gene revealed that it spans more than 18 kb and contains the coding sequence on 7 exons. The gene was mapped to chromosome 6 in a region with known homology to human chromosome 3q21.